Cystathionine β-synthase (CBS) plays an essential role in homocysteine (Hcy) metabolism in eukaryotes (Mudd et al., 2001, in The Metabolic and Molecular Bases of Inherited Disease, 8 Ed., pp. 2007-2056, McGraw-Hill, New York). The CBS enzyme catalyzes a pyridoxal 5′-phosphate (PLP; Vitamin B6)-dependent condensation of serine and homocysteine to form cystathionine, which is then used to produce cysteine by another PLP-dependent enzyme, cystathionine γ-lyase. In mammalian cells that possess the transsulfuration pathway, CBS occupies a key regulatory position between the remethylation of Hcy to methionine or its alternative use in the biosynthesis of cysteine. The relative flux between these two competing pathways is roughly equal and is controlled by intracellular S-adenosylmethionine (AdoMet) concentrations (Finkelstein and Martin, 1984, J. Biol. Chem. 259:9508-13). AdoMet activates the mammalian CBS enzyme by as much as 5-fold with an apparent dissociation constant of 15 μM (Finkelstein et al., 1975, Biochem. Biophys. Res. Commun. 66: 81-87; Roper et al., 1992, Arch. Biochem. Biophys. 298: 514-521; Kozich et al., 1992, Hum. Mutation 1: 113-123).
The C-terminal regulatory domain of human CBS consists of ˜140 amino acid residues (Kery et al., 1998, Arch. Biochem. Biophys. 355: 222-232). This region is required for tetramerization of the human enzyme and AdoMet activation (Kery et al., 1998, id.). The C-terminal regulatory region also encompasses the previously defined “CBS domains” (Bateman, 1997, Trends Biochem. Sci. 22: 12-13). These hydrophobic sequences (CBS 1 and CBS 2), spanning amino acid residues 416-468 and 486-543 of SEQ ID NO: 1, respectively, are conserved in a wide range of otherwise unrelated proteins. Their function remains unknown, although the sharp transition of thermally induced CBS activation and the observation that mutations in this domain can constitutively activate the enzyme indicates that they play a role in the autoinhibitory function of the C-terminal region (Janosik et al., 2001, Biochemistry 40: 10625-33; Shan et al., 2001, Hum. Mol. Genet. 10: 635-643; Miles and Kraus, 2004, J. Biol. Chem. 279: 29871-4). Two well-conserved CBS domains are also present in the C-terminal region of the yeast CBS, which is of approximately the same length as the human enzyme.
In healthy normal individuals, CBS-mediated conversion of Hcy to cystathionine is the rate-limiting intermediate step of methionine (Met) metabolism to cysteine (Cys). Vitamin B6 is an essential coenzyme for this process. In patients with certain genetic mutations in the CBS enzyme, the conversion of Hcy to cystathionine is slowed or absent, resulting in elevations in the serum concentrations of the enzymatic substrate (Hcy) and a corresponding decrease in the serum concentrations of the enzymatic product (cystathionine). The clinical condition of an elevated serum level of Hcy, and its concomitant excretion into the urine, is collectively known as homocystinuria.
Deficiency of CBS is the most common cause of inherited homocystinuria, a serious life-threatening disease that results in severely elevated homocysteine levels in plasma, tissues and urine. Estimates on the prevalence of homocystinuria vary widely. Ascertainment by newborn screening and clinical ascertainment have indicated a prevalence ranging from 1:200,000 to 1:335,000 (Mudd et al., 1995, The Metabolic and Molecular Basis of Inherited Diseases, McGraw-Hill: New York, p. 1279). The primary health problems associated with CBS-deficient homocystinuria (CBSDH) include: cardiovascular disease with a predisposition to thrombosis, resulting in a high rate of mortality in untreated and partially treated patients; connective tissue problems affecting the ocular system with progressive myopia and lens dislocation; connective tissue problems affecting the skeleton characterized by marfanoid habitus, osteoporosis, and scoliosis; and central nervous system problems, including mental retardation and seizures. Symptoms include dislocated optic lenses, skeletal disorders, mental retardation and premature arteriosclerosis and thrombosis (Mudd et al., 2001, id.). Homozygous CBS deficiency is associated with a multitude of clinical symptoms, including mental retardation, osteoporosis, kyphoscoliosis, stroke, myocardial infarction, ectopia lentis, and pulmonary embolism. Cardiovascular complications of the disease, in particular arterial and venous thrombosis, are the principal contributors to early mortality.
The pathophysiology of CBS deficiency is undoubtedly complex, but there is a consensus that the fundamental instigator of end-organ injury is an extreme elevation of serum Hcy, a substrate of CBS that builds-up in tissues and blood due to the absence of its CBS-catalyzed condensation with L-serine to form cystathionine. The toxicity of profound elevations in blood and tissue concentrations of Hcy may ensue from the molecular reactivity and biological effects of Hcy per se or from its metabolites (e.g. Hcy-thiolactone) that affect a number of biological processes (Jakubowski et al., 2008, FASEB J 22: 4071-6). Abnormalities in chronic platelet aggregation, changes in vascular parameters, and endothelial dysfunction have all been described in patients with homocystinuria.
Currently, three treatment options exist for the treatment of CBSDH:                1) Increase of residual activity of CBS activity using pharmacologic doses of Vitamin B6 in Vitamin B6-responsive patients        2) Lowering of serum Hcy by a diet with a strict restriction of the intake of Met; and        3) Detoxification by betaine-mediated conversion of Hcy into Met, thus lowering serum Hcy concentration.        
Each of these three therapies is aimed at lowering serum Hcy concentration. The standard treatment for individuals affected with Vitamin B6 non-responsive CBSDH consists of a Met-restricted diet supplemented with a metabolic formula and Cys in the form of cysteine (which has become a conditionally essential amino acid in this condition). Intake of meat, dairy products, and other food high in natural protein is prohibited. Daily consumption of a poorly palatable, synthetic metabolic formula containing amino acids and micronutrients is required to prevent secondary malnutrition. Supplementation with betaine (trade name: Cystadane™, synonym: trimethylglycine) is also standard therapy, wherein betaine serves as a methyl donor for the remethylation of Hcy to Met catalyzed by betaine-homocysteine methyltransferase in the liver (Wilcken et al., 1983, N. Engl. J. Med. 309: 448-53). Dietary compliance generally has been poor, even in those medical centers where optimal care and resources are provided, and this non-compliance has major implications on the development of life-threatening complications of homocystinuria.
To enable patients with homocystinuria enjoy a far less restrictive diet (e.g. daily intake limited to 2 g protein per kg, which is easily attainable), and have a significantly decreased Hcy plasma level leading in the long-term to clinical improvement, a strategy for increasing enzyme activity provides potential for treatment as set forth in co-pending U.S. provisional patent application Ser. No. 61/758,138. The most effective therapeutic strategy is to increase enzyme activity, as is evident when Vitamin B6-responsive homocystinuria patients are given pyridoxone. However, this strategy is not possible for Vitamin B6 non-responsive patients due to the nature of the mutations. Enzyme replacement therapy (ERT) as a way to increase enzyme activity in these patients requires exogenous enzyme, which is not present in the art and thus raises a need in the art for improved reagents and methods for producing CBS in greater yields of sufficiently purified enzyme for therapeutic administration.
Kraus and colleagues have developed expression systems and fermentation conditions for generating active recombinant human CBS and variants thereof (U.S. Pat. Nos. 5,635,375, 5,523,225 and 7,485,307, incorporated by reference herein in their entireties for any purpose). These proteins were purified by processes relevant for academic purposes, including use of protein leads on the proteins which are not considered useful for preparation of pharmaceuticals.
In order to employ methods of increasing CBS enzyme activity, an efficient method of CBS enzyme purification is required. Existing methods of purification for recombinant CBS protein rely on affinity tags to facilitate purification that does not provide the desired purity and efficiency. Therefore to more efficiently obtain the necessary levels of CBS required for therapeutic use there is a need for improved downstream purification of CBS protein produced in microbial cells.